Even before its brain has fully developed, a growing fruit fly-like most other creatures—has many important decisions to make. How many sets of legs? Where does the nervous system go?

Fortunately, the choices don't require thought. Instead, they are made by a collection of proteins whose job is to map out the body's blueprint. A recent study led by Dr. Susan Parkhurst's lab in the Basic Sciences Division now provides new insight into how one of these body-plan architects—a fruit-fly protein called Hairy-does its job.

So named because flies that lack it develop too many bristles, Hairy is one of a large family of proteins with counterparts in virtually all animals, including humans.

The new study is the first to identify the nearly five dozen genes that Hairy must shut off for the fly to assume its characteristic physique. The identity of these genes suggests that Hairy is responsible for a key cellular decision that occurs in cells of the developing fruit-fly embryo—whether to keep dividing aimlessly or to specialize into a dedicated type of tissue.

The findings will help scientists better understand the complex process of animal development and may shed light on analogous systems in humans, where missteps can lead to birth defects and the onset of cancers.

The results are published in the July issue of the Public Library of Science, a new journal dedicated to providing open access of research papers for the scientific community. Co-authors included Daniella Bianchi-Frias, a former research technician in Parkhurst's lab who is now a Molecular and Cellular Biology graduate student; Dr. Amir Orian, a postdoc in Dr. Bob Eisenman's lab; Dr. Jeff Delrow, head of the Genomics shared resource; Dr. Julio Vazquez, manager of the Scientific Imaging shared resource; and Alicia Rosales-Nieves, a research technician in Parkhurst's lab.

Scientists have searched for the genes controlled by Hairy for years with little success, Parkhurst said.

"Hairy acts at multiple times during development of the fly," she said. "There simply wasn't a systematic way to look for all of its target genes."

What made it possible now, she said, was the collective effort of Fred Hutchinson collaborators who have developed methods to identify gene targets of proteins like Hairy.

Hairy is a repressor, a type of protein that shuts off genes when it binds to DNA. Hairy is one of the first genes to act in the developmental circuitry of the fly when the embryo establishes the characteristic segments of the insect's body. Although most repressors work by binding to DNA near the genes they control, Hairy is known as a long-range repressor, meaning that it can shut off genes even when it is bound to DNA at some distance from its target.

DamID method

To identify genes controlled by Hairy, Parkhurst's lab first used a method called DamID, which was originally developed in Dr. Steven Henikoff's lab. The technique enables scientists to find genes that come into close physical contact with a protein of interest, in this case Hairy. The researchers then used microarrays—glass slides containing tiny droplets of DNA that allowed the researchers to scan through thousands of fly genes simultaneously— to determine the identity of the genes. Delrow and colleagues in the Genomics shared resource produced the microarrays and developed the strategies used to interpret the data.

The experiment revealed 59 genes that appeared to be under Hairy's control. Yet Parkhurst wanted additional evidence that the target genes were in fact the right ones because Hairy binds DNA relatively far away from the genes it regulates, but the DamID technique works best at identifying target genes in proximity to DNA-bound proteins. To obtain the proof she needed, Parkhurst turned to Vazquez for help with a technique that would allow her to actually see, using a microscope, the Hairy protein bound near the 59 genes on fly chromosomes.

Although most chromosomes are too small to be analyzed in this way, the salivary glands of fruit flies contain greatly enlarged chromosomes—called polytene chromosomes—that are easy to view microscopically. Using methods that allowed the Hairy protein to be tagged with a color to stand out against the background of the chromosomes, Vazquez was able to verify that Hairy bound to all of the 59 genes identified by the DamID technique.

Hairy in central control

Parkhurst said that the target genes fell into distinct classes. In addition to genes involved in defining the segments that make up the insect body, of particular interest was the finding that Hairy also regulates genes involved in cell division, cell growth or cell shape. Changes in cell shape accompany a cell's commitment to adopt a dedicated fate, such as becoming muscle or nervous tissue.

"A cell can't grow and divide while it is changing its shape," Parkhurst said. "Our results suggest that Hairy is controlling how the cell decides when it has grown enough and it's time to differentiate, which is something that had never really been appreciated. It means that Hairy is sitting in a central place to coordinate the early events of development."

The family of repressor proteins to which Hairy belongs is often involved in developmental "switch" decisions: They regulate the choice between alternate pathways and bring about changes in cell fate. Mistakes in these regulatory steps often lead to developmental defects and the onset of leukemias or other cancers. The numerous processes requiring Hairy-family proteins and the techniques available for study in the fruit fly make it an excellent organism to probe the functions of these proteins in a living animal. Results obtained from these projects are expected to have wide implications as Hairy family proteins and mechanisms of repression are similar among all organisms.

Next, Parkhurst's lab plans to study the biological functions of some of the Hairy target genes in more detail to see how they help Hairy coordinate distinct developmental events.